Cage-like bifunctional chelators, copper-64 radiopharmaceuticals and PET imaging using the same

ABSTRACT

Disclosed is a class of versatile Sarcophagine based bifunctional chelators (BFCs) containing a hexa-aza cage for labeling with metals having either imaging, therapeutic or contrast applications radiolabeling and one or more linkers (A) and (B). The compounds have the general formula 
     
       
         
         
             
             
         
       
         
         
           
             where A is a functional group selected from group consisting of an amine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and an alkene, and B is a functional group selected from the group consisting of hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, a thiol, an azide and an alkene. Also disclosed are conjugate of the BFC and a targeting moiety, which may be a peptide or antibody. Also disclosed are metal complexes of the BFC/targeting moiety conjugates that are useful as radiopharmaceuticals, imaging agents or contrast agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.12/695,125 filed Jan. 27, 2010, which claims the benefit of U.S.Provisional Application No. 61/147,709, filed Jan. 27, 2009, the entirecontents of all of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This work was supported by research grant from the Department of Energy.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Molecular imaging is an emerging technology that allows forvisualization of interactions between molecular probes and biologicaltargets. Positron emission tomography (PET), micro-PET and PET/CT, arestate-of-the-art nuclear medicine imaging modalities, which use nano- topicomolar concentrations of the corresponding probes (radiotracers) toachieve images of biological processes within the living system.Selection of the proper radionuclide and synthetic approach forradiotracer design are critical. Positron-emitting isotopes frequentlyused include ¹¹C and ¹⁸F. One non-traditional PET radionuclide, ⁶⁴Cu,shows promise as both a suitable PET imaging and therapeuticradionuclide due to its nuclear characteristics (T_(1/2)=12.7 h, β⁺+:17.4%, E_(β+max)=656 keV; β⁻: 39%, E_(β−max)=573 keV), and theavailability of its large-scale production with high specific activity.

Stable attachment of radioactive ⁶⁴Cu²⁺ to targeted imaging probesrequires the use of a bifunctional chelator (BFC), which is used toconnect a radionuclide and bioactive molecule to form the⁶⁴Cu-radiopharmaceutical. Extensive efforts have been devoted to thedevelopment of BFCs for ⁶⁴Cu labeling. Three of the most commonchelators studied have been the macrocyclic ligands DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid), TETA(1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N″′-tetraacetic acid), andcross-bridged tetraamine ligands. (Refs. 1-3) However, dissociation of⁶⁴Cu from the BFC in vivo and harsh labeling conditions (e.g.,incubation at 75° C. under basic conditions) impair the use of thesechelators in preparing biomolecule-based ⁶⁴Cu-radiopharmaceuticals.(Ref. 4) The BFC DOTA has been used for ⁶⁴Cu²⁺ labeling. (Refs. 3, 5, 6)However, the limited stability of the copper chelate in vivo hashindered its application. (Ref. 37)

The integrin α_(v)β₃ receptor has been the attractive target ofintensive research given its major role in several distinct processes,such as tumor angiogenesis and metastasis, and osteoclast mediated boneresorption. (Refs. 7, 8) The molecular imaging of integrin α_(v)β₃expression will allow the detection of cancer and other angiogenesisrelated diseases, patient stratification, and treatment monitoring ofanti-angiogenesis based therapy. (Refs. 9, 10) Although we and othershave successfully developed various DOTA conjugated RGD peptides formultimodality imaging of integrin α_(v)β₃ expression. (Refs. 9, 10, 11,31), the loss of ⁶⁴Cu from the chelator has lead to unfavorable highretention in liver, resulting in high background. Therefore, the choiceof a more stable BFC is preferred.

Although some other novel BFCs have been developed and shown promise foruse in copper-64 labeling (Refs. 2, 4, 12, 32), there is lack ofadequate published data regarding the biological activity of thesecomplexes. Some have high uptake in lung, liver and muscle, which mayimpair the detection of small lesions in the chest or/and abdominalregions. (Refs. 2, 4, 12, 32)

Recently, Sargeson and co-workers reported a new type BFC based on thesarcophagine (3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6] icosane, Sar,FIG. 1.A-2) for preparation of ⁶⁴Cu-radiopharmaceuticals, (Refs. 2, 36)These ligands coordinate ⁶⁴Cu²⁺ within the multiple macrocyclic ringscomprising the Sar cage structure, yielding stable complexes that areinert to the dissociation of the metal ion.

The caged-like BFC Sar ligands are able to selectively label ⁶⁴Cu²⁺rapidly over a wider range of pH value under mild conditions. (Ref. 2)However, there are only a few reports that describe the complexation,stability and biodistribution of the ⁶⁴Cu complexes of the Sar ligand,and only (NH₂)₂-Sar (Diamsar) and 1-N-(4-aminobenzyl)-3, 6, 10, 13, 16,19-hexaazabicyclo [6.6.6] icosane-1, 8-diamine (SarAr) have beenreported as a BFC for the development of ⁶⁴Cu-radiopharmaceuticals.(Refs. 2, 13, 35, 36) Moreover, the relatively nontrivial and multi-stepsynthesis of Sar ligands may limit their future applications. (Ref. 4)However, the SARAR BFC utilizes the C-terminal for carboxylic acid forconjugation. This may be a disadvantage as the C-terminus is often foundto be a crucial part for maintaining biological activity.

As such, there is a need for improved bifunctional chelators that canefficiently form stable complexes with metals, including Copper-64 undermild conditions.

There is also a need for new radiopharmaceuticals and imaging agentsthat are stable in vivo for imaging and other.

There is also a need for simplified methods for making bifunctionalchelators.

BRIEF SUMMARY OF THE INVENTION

It is one object of the present invention to provide improvedbifunctional chelators (BFCs) that form stable complexes with metals,including Copper-64, that are stable in vivo.

It is another object of the present invention to provide BFC/TargetingMoiety conjugates that specifically target biomolecules.

It is another object of the present invention to provide BFC/TargetingMoiety conjugate complexed with metals useful as imaging, therapeutic orcontrast agents.

One embodiment of the present invention is directed to bifunctionalchelators having the general formula

where A is a functional group selected from group consisting of anamine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and analkene, and B is a functional group selected from the group consistingof hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, athiol, an azide and an alkene. In one preferred embodiment, A is acarboxylic acid, or a salt or ester thereof. Even more preferably, A isa carboxylic acid having the formula

and wherein R₁, R₂, R₃, and R₄ are independently selected from the groupconsisting of H, halogen, alkyl, alkoxy or alkene, or a salt or esterthereof. In a preferred embodiment, R₁, R₂, R₃, and R₄ are hydrogen.

In another preferred embodiment, the bifunctional chelators are

or a salt or ester thereof.

Another embodiment of the present invention is directed to bifunctionalchelator/Targeting Moiety conjugates in which a bifunctional chelator isconjugated to a targeting moiety. In the conjugate, the bifunctionalchelator is

where A is a functional group selected from group consisting of anamine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and analkene, and B is a functional group selected from the group consistingof hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, athiol, an azide and an alkene, or a salt or ester thereof. In apreferred embodiment, the targeting moiety is selected from the groupconsisting of a peptide and antibody. For instance, the targeting moietymay be a peptide selected from the group consisting of a RGD,Asp-Gly-Glu-Ala (DGEA) (SEQ ID NO: 1), bombesin peptides (BBN), uPARpeptides, and other peptides with a lysine amine or N-terminal.

Another embodiment of the present invention is directed to a kit havinga bifunctional chelator/Targeting Moiety conjugate with instructions forcomplexing a metal to the bifunctional chelator/Targeting Moietyconjugate.

The present invention is also directed new compositions comprising abifunctional chelator conjugated to a Targeting moiety and complexedwith a metal. These new compositions are useful as radiopharmaceuticals,therapeutic agents or contrast agents. In these compositions, thebifunctional chelator is

where A is a functional group selected from group consisting of anamine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and analkene, and B is a functional group selected from the group consistingof hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, athiol, an azide and an alkene, or a salt or ester thereof. In thesecomplexes, metal resides within the Sar cage and is bonded to one ormore nitrogen. Preferably, the metal may be of ⁹⁰Y, Gd, ⁶⁸Ga, ⁵⁷Co,⁶⁰Co, ⁵²Fe, ⁶⁴Cu or ⁶⁷Cu.

In one preferred embodiment, the present application is directed to aradiopharmaceutical comprising ⁶⁴Cu-AmBaSar-RGD, either alone ortogether with a carrier.

DESCRIPTION OF THE FIGURES

FIG. 1 is a chart depicting a procedure for the synthesis of thebifunctional chelator AmBaSar

FIG. 2 is a chart depicting a procedure for the synthesis of AmBaSar-RGDand labeled with Copper-64.

FIG. 3 is a representative ¹H-NMR spectra in D₂O of AmBaSar.

FIG. 4 is a representative HPLC chromatogram of AmBaSar.

FIG. 5 a representative chromatogram of crude AmBaSar-RGD using thesemipreparative HPLC system.

FIG. 6 is a representative mass spectra of AmBaSar-RGD

FIG. 7 is representative radio-HPLC chromatogram of crude⁶⁴Cu-AmBaSar-RGD using the analytical HPLC system described herein.

FIG. 8 shows a synthesis scheme for an improved method of synthesizingthe bifunctional chelator AmBaSar.

FIG. 9 is a chart depicting a method for the radiosynthesis of⁶⁴Cu-AmBaSar.

FIG. 10 is a representative chromatogram of crude ⁶⁴Cu-AmBaSar usinganalytical HPLC system

FIG. 11 is a graph showing the in vitro stability of ⁶⁴Cu-AmBaSar in PBS(pH 7.4) and FBS at 37° C.

FIG. 12 shows coronal sections of a microPET study of Balb/c mouse aftersingle intravenous of ⁶⁴Cu-AmBaSar and ⁶⁴Cu-DOTA at 30 min and 2 hour.K=Kidney; L=Liver; B=Bladder.

FIG. 13 is a graph showing the biodistribution of ⁶⁴Cu-AmBaSar and⁶⁴Cu-DOTA in Balb/C mice after 2 h postinjection.

FIG. 14 is a chart comparing the chemical structures of ⁶⁴Cu-AmBaSar-RGDand ⁶⁴Cu-DOTA-RGD.

FIG. 15 is a graph showing the in vitro stability of ⁶⁴Cu-AmBaSar-RGD inPBS (pH 7.4), FBS, and mouse serum for incubation under 37° C. for 3,18, and 24 h.

FIG. 16 is a graph showing that the U87MG cell uptakes of⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD at 0.5, 1, and 2 h in theabsence/presence of excess amount of cyclic RGD

FIG. 17 is a MicroPET study of U87MG tumor-bearing mice showing: (A) Thecoronal images of nude mice bearing U87MG tumor at 1, 2, 4, and 20 hp.i. of ⁶⁴Cu-AmbaSar-RGD and ⁶⁴Cu-DOTA-RGD; and (B) Time activity curvesof ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD in U87MG tumor, liver, and kidneys(n=3). Tumors are indicated by arrows.

FIG. 18 is a MicroPET imaging study of ⁶⁴Cu-AmBaSar-RGD and⁶⁴Cu-DOTA-RGD on U87MG xenograft mouse model at 2 h p.i. with/without ablocking dose of cyclic RGD: (A) microPET coronal images; (B).Quantitative analyses of microPET imaging of U87MG tumor, muscle, liver,and kidneys (n=3). Arrows indicate the tumor positions.

FIG. 19 shows (A) a graph showing biodistribution data for⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD in mice bearing U87MG gliomaxenografts (mean±SD, n=3) at 20 h p.i.; and (B) a graph showing theratios of tumor to main organs uptake of ⁶⁴Cu-AmBaSar-RGD and⁶⁴Cu-DOTA-RGD.

FIG. 20A and FIG. 20B. FIG. 20A, Metabolic stability of ⁶⁴Cu-AmBaSar-RGDand ⁶⁴Cu-DOTA-RGD in mouse blood sample and in liver, kidney and U87 MGtumor homogenates at 1 h post injection. FIG. 20B, The HPLC profiles ofpure ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD (Standard) are also shown.

FIG. 21 is a chart showing a synthetic method for the preparation ofthiol and carboxylate functionalized BFCs according to the presentinvention.

FIG. 22 is a chart showing a synthetic method for the preparation ofDiBaSar according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is directed to a class ofversatile Sarcophagine based bifunctional chelators containing ahexa-aza cage for labeling with metals having either imaging,therapeutic or contrast applications radiolabeling and one or morelinkers (A) and (B). The compounds of the present invention have thegeneral formula

where A is a functional group selected from group consisting of anamine, a carboxylic acid, an ester, a carbonyl, a thiol, an azide and analkene, and B is a functional group selected from the group consistingof hydrogen, an amine, a carboxylic acid, and ester, a carbonyl, athiol, an azide and an alkene. Suitable linker group A and B should beof sufficient length (˜6 atom lengths) and should incorporate a reactivegroup that can be readily attached to a range of target agents, but notinterfere with efficient and selective complexation of a metal ion likeCu²⁺ to the sarcophagine cage, as well as the target agent's biologicalactivity. (Ref. 16)

In one embodiment of the present invention, linkers A or B, or both, maypreferably be a carboxylic acid of the following formula:

Where R₁, R₂, R₃, and R₄ are independently selected from the groupconsisting of H, halogen, alkyl, alkoxy or alkene.

In another embodiment of the present invention, the bifunctionalchelators are sarcophagine based compositions, referred to herein asAmBaSar and DiBaSar, having the having the following structure:

The bifunctional chelators may be conjugated to a targeting moiety asBFC/Targeting Moiety conjugates that specifically target a range ofbiological molecules. In two separate embodiments, the targeting moietyis a peptide or an antibody.

For instance, the pendant carboxylate group of carboxylic acid (ROOH)functionalized BFC, including AmBaSar and DiBaSar, can be directlyconjugated to the amine of lysine in biological molecules, which permitthe development of new biomolecule-based ⁶⁴Cu-radiopharmaceuticals. Morespecifically, the BFC's can be used to directly conjugate targetingpeptides (Z) through the formation of an amide bond with the peptide toform BFC peptide conjugates of the following structure:

where Z is a targeting peptide containing lysine.

Examples of the peptides to which AmBaSar, and other carboxylic acidbased BFCs may be conjugated include, but are not limited to, the smallcyclic peptide Arg-Gly-Asp (RGD). Asp-Gly-Glu-Ala (DGEA) (SEQ ID NO: 1),bombesin peptides (BBN), uPAR peptides, and other peptides with a lysineamine or N-terminal. Sar-DGEA is directly conjugated to the Sar cagewithout functionalization by solid or liquid phase methods well known inthe art. In vitro and in vivo evaluation of the AmBaSar-RGD demonstratesthe potential of the BFC's of this invention for preparation of a rangeof radiopharmaceuticals.

The BFC and the BFC/Targeting Moiety conjugates of the present inventionmay be complexed with a range of metals by known methods to produceradiopharmaceuticals for imaging or therapy. The use of the word “metal”or “metals” throughout this specification should be understood toinclude metal ions and their salts. The BFC and the BFC/Targeting Moietyconjugates may also be combined with paramagnetic metals for use ascontrast agents in MR imaging or with these or other metals for CTscanning applications. Examples of suitable metal ions for use in thepresent invention include, but are not limited to, ⁹⁰Y, Gd, ⁶⁸Ga,^(57/60)Co, ⁵²Fe, ^(64/67)Cu In the Metal-BFC and Metal-BFC/TargetingMoeity complexes of the present invention, the metal resides within theSar cage bound to the nitrogen of the Sar Cage as shown in FIG. 14 for⁶⁴Cu-AmBaSar-RGD.

In order to form the radiopharmaceuticals in accordance with the presentinvention, the BFC/Targeting Moiety conjugates complexed with a metalmay be dispersed or dissolved in a suitable carrier in appropriateconcentrations and delivered to the patient in appropriate doses.Preferred routes of delivery are oral, injection, or infusion. Theidentity of the carrier is not particularly limited so long as theradiopharmaceutical is stable in the carrier. Suitable carriers for usein connection with the present invention include Saline (0.9%),Phosphate Buffer Solution, or Ethanol solution (<8%). The carrier mayoptionally include HSA protein, synthetic polymers, dendrimers,cyclodextron et al. The active species should be delivered in an amounteffective to produce the desired effect but below the level where thereis unacceptable toxicity. When used in solution, concentration of activespecies in the carrier should be sufficiently high to produce thedesired effect but below the level where there is significant toxicity.Typical concentrations for imaging applications are approximately in therange of 1-20 mCi/mL.

In one example of the present invention, AmBaSar is conjugated to thesmall cyclic Arg-Gly-Asp (RGD) peptide, and subsequently labeled with⁶⁴Cu, to provide a new PET probe ⁶⁴Cu-AmBaSar-RGD for imaging theα_(v)β₃ integrin receptor. ⁶⁴Cu-AmBaSar-RGD has the following formula:

Another embodiment of the present invention is directed to newradiopharmaceuticals comprising ⁶⁴Cu-AmBaSar-RGD. ⁶⁴Cu-AmBaSar-RGD isobtained with high radiochemical yield (≥95%) and purity (≥99%) undermild conditions (pH 5.0˜5.5, 23˜37° C.) in less than 30 min. In order toform the radiopharmaceuticals in accordance with the present invention,⁶⁴Cu-AmBaSar-RGD may be dispersed or dissolved in a suitable carrier inappropriate concentrations delivered to the patient in appropriate dosesorally, or by injection, infusion or, where possible. Suitable carriersfor use in connection with ⁶⁴Cu-AmBaSar-RGD include Saline (0.9%),Phosphate Buffer Solution, or Ethanol solution (<8%). Carrier mayinclude HSA protein, synthetic polymers, dendrimers, cyclodextron et al.The concentration of ⁶⁴Cu-AmBaSar-RGD in the carrier should besufficiently high to produce sufficient signal to permit tracking and/orimaging of the ⁶⁴Cu-AmBaSar-RGD within the patient after deliver butbelow the level where there is significant toxicity. Suitableconcentrations for the ⁶⁴Cu-AmBaSar-RGD are preferably 1˜20 mCi/mL.

In another embodiment of the present invention, a kit comprises aBFC/Targeting Moiety conjugate together with instructions for formingthe metal complex with the BFC/Targeting Moiety conjugate.

The BFC, BFC/Targeting Moiety and the BFC/Targeting Moiety conjugatecomplexed with a metal may each be in the form of a pharmaceuticallyacceptable salts or ester form. Any carboxylic acid described herein isalso understood to encompass pharmaceutically salt or esters of thecarboxylic acid.

Metabolic studies support the observation that ⁶⁴Cu-AmBaSar-RGD is morestable in vivo than ⁶⁴Cu-DOTA-RGD. In vitro and in vivo evaluations ofthe ⁶⁴Cu-AmBaSar-RGD demonstrate its improved stability compared withthe established tracer ⁶⁴Cu-DOTA-RGD. For in vitro studies, theradiochemical purity of ⁶⁴Cu-AmBaSar-RGD was more than 97% in the PBS orFBS and 95% in mouse serum after 24 hr incubation. The log P value of⁶⁴Cu-AmBaSar-RGD was −2.44±0.12. For in vivo studies, ⁶⁴Cu-AmBaSar-RGDand ⁶⁴Cu-DOTA-RGD have demonstrated comparable tumor uptake at selectedtime points based on microPET imaging. The integrin α_(v)β₃ receptorspecificity was confirmed by blocking experiments for both tracers.Compared with ⁶⁴Cu-DOTA-RGD, ⁶⁴Cu-AmBaSar-RGD exhibits much lower liveraccumulation in both microPET imaging and biodistribution studies.

Abbreviations and Nomenclature

The IUPAC names for the cage ligands of the present invention and metalcomplexes of the cage compounds are long and complicated. The names forthe ligands described or discussed herein are abbreviated as follows:

-   3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6] icosane is referred to    “sarcophagine”, or “Sar”;-   1, 8-Diamine-3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6] icosane is    referred to as “Diamsar”;-   1-N-(4-aminobenzyl)-3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6]    icosane-1, 8-diamine is referred to as “SarAr”;-   Methyl 4-((8-amino-3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6]    icosane-1-ylamino) methyl) benzoate is referred to as “AmMBSar”; and-   4-((8-amino-3, 6, 10, 13, 16, 19-hexaazabicyclo [6.6.6]    icosane-1-ylamino) methyl) benzoic acid is referred to as “AmBaSar”;-   1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid is    referred to as DOTA, and-   1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N″′-tetraacetic acid is    referred to as TETA.

Unless otherwise indicated, complexes described herein are named using anomenclature which represents these M-hexaamine cage complexes by “M-(A,Bsar)”, where M represents metal ion, and A and B are the substituentsin the 1- and 8-positions of the Sar cage:

-   (1, 8-Dinitro-Sar) cobalt(III) trichloride is represented by the    chemical formula [Co(DiNosar)]Cl₃;-   (1, 8-Diamine-Sar) cobalt(III) pentachloride is represented by the    chemical formula [Co(DiAmSar)]Cl₅;-   (1, 8-Diamine-Sar) copper(II) tetrachloride is represented by the    chemical formula [Cu(DiAmSar)]Cl₄;-   (1-amine, 8-(aminomethyl) 4′-methylbenzoate-Sar) copper(II) is    represented by the chemical formula [Cu(AmMBSar)]²⁺;-   (1-amine, 8-(aminomethyl)-benzoic acid-Sar) copper(II) is    represented by the chemical formula [Cu(AmBaSar)]²⁺;-   (1, 8-(aminomethyl) 4′-methylbenzoate-Sar) copper(II) is    [Cu(DiAMBSar)]²⁺.    Design, Synthesis, and Characterization of Bifunctional Chelator    AmBaSar

In order to design a suitable bifunctional chelator for ⁶⁴Cu labelingand conjugating with RGD, it was necessary to modify the linkage ofdiamsar. The aromatic amine of SarAr can be replaced with an aromaticcarboxyl, which can conjugate a cyclic peptide containing the RGD motifin addition to a lysine for conjugating to the chelator, to form BFCAmBaSar. AmBaSar can efficiently label ⁶⁴Cu²⁺ due to the provision of athree-dimensional hexa-aza cage which increases thermodynamic andkinetic stability to complex ⁶⁴Cu²⁺ or other metal ions, while allowingthe aromatic linker with carboxyl acid group to conjugate with the amineof lysine in the cyclic peptide containing the RGD motif. TheAmBaSar-RGD can be used to form new nuclear, MRI, and optical probes bycomplexion with other appropriate metal radioisotopes or paramagneticmetal ions using similar preparations. Appropriate metals include, butare not limited to ⁹⁰Y, Gd, ⁶⁸Ga, ^(57/60)Co, ⁵²Fe and ^(64/67)Cu.

AmBaSar can be reacted with a variety of peptides or biomolecules, sincethe chemistry for conjugating AmBaSar to other peptides is similar tothat described for the cyclic peptide RGD. The identity of the peptidesis not particularly limited as long as pendant carboxylate group orcarboxylic acid group of the AmBaSar chelator be directly conjugated toan amine of a lysine or N-terminal in the peptide. Preferably however,the peptide is to which AmBaSar is reacted is one that targets aspecific biomolecule. Other peptides include, but are not limited to,Asp-Gly-Glu-Ala (DGEA) (SEQ ID NO: 1), bombesin peptides (BBN), uPARpeptides, and other peptides with a lysine amine or N-terminal.

An efficient synthesis of AmBaSar is shown in FIG. 1. The degree ofhydration of the synthesized compounds is dependent on the method ofpurification. FIG. 1, Compounds 1˜4 may be prepared based on methodsdescribed in the literature using cobalt complexes as startingmaterials, (Refs. 14, 15) However, the reported syntheses of compound1˜4 are incomplete and with compounds not fully characterized. Thecomprehensive synthetic methods and full characterization of thesecompounds are disclosed herein.

In brief, and as shown in FIG. 1-1, tris(ethylenediamine)cobalt(III)chloride initiates the encapsulation process using the reactivenucleophile nitromethane and formaldehyde in alkaline solution at roomtemperature to form a hexa aza cage containing Co(III) compound 1, whichwas then reduced with zinc dust in acid solution to give the very stableCo(III) diamsar complex 2. The Co(III) ion of compound 2 can be removedby reduction with high concentrations of hydrochloric or hydrobromicacid at high temperatures (130˜150° C.) or excess cyanide ion to yieldthe free cage. Here we used excess cyanide ion to remove the metal ionin compound 2 to form compound 3 in good yield (58.5%). The six nitrogendonor atoms of the hexa aza cage allows strong binding to many metalions, such as Cu²⁺, so compound 3 was easy to complex with metal ionCu²⁺, yielding compound 4 under weak acid conditions. The compounds 1˜4were characterized by elemental analysis, MS and ¹H-NMR, the results areconsistent with literature reports. (Refs. 14, 15.)

It is difficult to functionalize the apical primary amines of diamsar 3(FIG. 1) directly because there are 2 primary and 6 secondary amines indiamsar, which potentially could create many by-products and difficultyduring purification. The initial formation of copper(II) complex ofdiamsar 4 (FIG. 1) ties up the 6 secondary amines of diamsar, and alsopermit the tracking of the resultant Cu(II) complexes on the ionexchange columns. (Ref. 16.) Structural studies have confirmed that itis possible to exploit the relatively low nucleophilicity of the Cu(II)complex of diamsar in acylation and alkylation reactions leading to avariety of functionalized cage amine complexes. (Refs. 17, 18). Thehydride reducing agent sodium cyanoborohydride (NaBH₃CN) is used forreduction due to its stability in relatively strong acid solutions(about pH 3), its solubility in hydroxylic solvents such as ethanol, andits different selectivities at different pH values. At pH 3˜4 it reducesthe imine to an amine efficiently, while this reduction becomes veryslow at higher pH values. (Ref. 19) The reducing reaction of the cageamine of compound 4 with aromatic methyl 4-formylbenzoate under NaBH₃CNethanol acid solution yields aromatic functionalization of the cageamine compound 5 and the by-product bis-4-formylbenzoate diamsarcomplex. This reaction took a significant amount of time (4 days) andyield was 27.9% due to low nucleophilicity. Separation was carried outby ion exchange chromatography. Compound 6 was achieved by demetallationof compound 5, followed by alkaline hydrolysis to produce the targetcompound 7 AmBaSar according to the reported method. (Ref. 20)

AmBaSar Conjugating Cyclic Peptide RGD and ⁶⁴Cu Radiolabeling:

AmBaSar contains one carboxyl group and an inactive primary amine, whichmeans that it is a mono-functional molecule that can react with a cyclicpeptide containing the RGD motif in addition to a lysine for conjugatingto the chelator. Literature methods may be used for conjugation of thecyclic RGD with AmBaSar and preparing ⁶⁴Cu radiolabeled conjugates.(Refs. 6, 21, 22)

FIG. 2 is a chart showing the method for AmBaSar conjugating cyclic RGDand ⁶⁴Cu radiolabeling. The cyclic RGD conjugate AmBaSar-RGD wassynthesized in 80% yield and purified by semipreparative HPLC.Analytical HPLC found the retention time of AmBaSar-RGD to be 3.8 min,whereas cyclic RGD peptide eluted at 25 min under the same condition.AmBaSar-RGD was analyzed by mass spectrometry, found m/z=1049.3 for[M+H]⁺ (M=C₅₀H₈₀N₁₆O₉) and 1089.7 for [M+K]⁺.

In another embodiment of the present invention, the peptide-chelatorconjugate AmBaSar-RGD can be complexed with ⁶⁴Cu to form a new PETtracer for imaging the α_(v)β₃ integrin receptor. AmBaSar-RGD waslabeled with ⁶⁴Cu in 0.1 M ammonium acetate (pH 5.0) solution at roomtemperature (25° C.) for 1 h. The free ⁶⁴Cu-acetate was eluted at 3.2min, while ⁶⁴Cu-AmBaSar-RGD was eluted at 15.8 min by analytical HPLC,which was confirmed by cold Cu-AmBaSar-RGD. The radiochemical yieldobtained was ≥80% and the radiochemical purity was ≥95%.

EXPERIMENTAL

Methods and Materials:

¹H NMR spectra were obtained using a Varian Mercury 400 MHz instrument(USC NMR Instrumentation Facility), and the chemical shifts werereported in ppm on the δ scale relative to an internal TMS standard.Microanalyses for carbon, hydrogen, nitrogen and chlorine, cobalt,copper were carried out by the Columbia Analytical Services, Inc(Tucson, Ariz.). Mass spectra using LC-MS were operated by theProteomics Core Facility of the USC School of Pharmacy. Thin-layerchromatography (TLC) was performed on silica gel 60 F-254 plates(Sigma-Aldrich) using a mixture solution of 70% MeOH and 30% aqueousNH₄OAc (NH₄OAc solution is 20% by weight) as the mobile phase.Ion-exchange chromatography was performed under gravity flow using Dowex50WX2 (H⁺ form, 200-400 mesh) or SP Sephadex C25 (Na⁺ form, 200-400mesh) cation exchange resins. All evaporations were performed at reducedpressure (ca. 20 Torr) using a Büchi rotary evaporator.

HPLC was accomplished on two Waters 515 HPLC pumps, a Waters 2487absorbance UV detector and a Ludlum Model 2200 radioactivity detector,operated by Waters Empower 2 software. Purification of the conjugateAmBaSar-RGD was performed on a Phenomenex Luna C18 reversed phase column(5 μm, 250×10 mm); the flow was 3.2 mL/min, with the mobile phasesolvent A (12% acetonitrile in water), and the absorbance monitored at254 nm. The analytical HPLC was done on a Phenomenex Luna C18 reversedphase column (5 μm, 250×4.6 mm) and monitored using a radiodetector andUV at 218 nm. The flow was 1.0 mL/min, with the mobile phase startingfrom 99% solvent B (0.1% TFA in water) and 1% solvent C (0.1% TFA inacetonitrile) (01 min) to 70% solvent B (0.1% TFA in water) and 30%solvent C (0.1% TFA in acetonitrile) (1˜30 min).

All reagents and solvents were purchased from Sigma-Aldrich Chemicals(St. Louis, Mo., USA) and used without further purification unlessotherwise stated. N-hydroxysulfosuccinimide sodium salts (SNHS) andN-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl)were obtained from the Sigma-Aldrich Chemical Co. The cyclic RGDyKpeptide was purchased from Peptides International, Inc (Louisville, Ky.,USA). ⁶⁴CuCl₂ was purchased from MDS Nordion (Vancouver, BC, Canada).Water was purified using a Milli-Q ultra-pure water system fromMillipore Corp. (Milford, Mass., USA).

Experimental: AmBaSar Synthesis and Characterization Synthesis of[Co(DiNoSar)]Cl₃ (1)

To a solution of tris(ethylenediamine)cobalt(III) chloride dehydrate(1.2 g, 3.0 mmol) in water (4.0 mL) was added nitromethane (0.7 g, 12mmol) and aqueous formaldehyde (37%, 2.4 g, 30 mmol). The resultingsolution was cooled to 4° C. on an ice-water bath. Aqueous NaOH (4.0 M,3.0 mL) was cooled to 4° C. and mixed with the resulting solution above.The combined solution was stirred on ice-water bath where it rapidlyturned deep violet-brown from the initially orange color, and thereaction temperature was allowed to raise to room temperature 23-25° C.After 90 min, the reaction was quenched by the addition of HCl (6.0 M,2.0 mL). The orange precipitate formed was collected by filtration aftercooling on ice for 2 h, washed with methanol, and dried to provide 1(1.46 g, 90.2%). ¹H-NMR (D₂O): δ 3.55-3.65 (d, 6H, NCCH₂N); 3.15-3.35(d, 6H, NCCH₂N); 3.00-3.10 (d, 6H, NCH₂CH₂N); 2.55-2.65 (d, 6H,NCH₂CH₂N). MS: Calcd for C₁₄H₃₁CoN₈O₄[M+1-3HCl]⁺ m/z 431.2, found 431.5.Elemental analysis calculated for C₁₄H₃₀Cl₃CoN₈O₄, requires C, 31.15; H,5.60; N, 20.76; Cl, 19.71; Co, 10.92. Found: C, 30.90; H, 5.46; N,20.13; Cl, 20.80; Co, 10.70.

Synthesis of [Co(DiAmSar)]Cl₅.H₂O (2)

[Co(DiNoSar)]Cl₃ (2.0 g, 3.7 mmol) was dissolved in deoxygenated water(100 mL) under N₂ atmosphere. Zinc dust (2.3 g, 35 mmol) was added intothe solution with stirring for 5 min, followed by addition of HCl (6 M,15 mL) dropwise. The resulting solution continued to stir for anadditional 60 min under N₂ atmosphere. The N₂ flow was stopped and 30%H₂O₂ (1.0 mL) was added. The resulting solution was warmed for 15 min on75° C. water bath, then cooled and placed on a Dowex 50WX2 cationexchange column and washed with water (120 mL), followed by HCl (1.0 M,120 mL). The complex was then eluted with HCl (3.0 M, 400 mL) and theyellow eluate was collected and dried under vacuum to yield 2 (1.78 g.87%). ¹H-NMR (D₂O): δ 3.30-3.10 (m, 12H, NCCH₂N); 2.50-2.65 (m, 12H,NCH₂CH₂N). MS Calcd for C₁₄H₃₅CoN₈ [M+1-5HCl-H₂O]³⁰ m/z 371.2, found371.7. Elemental analysis calculated for C₁₄H₃₈Cl₅CoN₈O, requires C,29.46; H, 6.71; N, 19.63; Cl, 31.06; Co, 10.33. Found: C, 29.06; H,6.73; N, 19.04; Cl, 25.30; Co, 9.50.

Synthesis of Diamsar.5H₂O (3)

Co(DiAmSar)]Cl₅.H₂O (3.58 g, 6.3 mmol), NaOH (0.58 g, 14.5 mmol,sufficient to neutralize the protonated primary amino groups) andCobalt(II) chloride hexahydrate (1.6 g, 6.4 mmol) were dissolved indeoxygenated water (50 mL) under nitrogen. Sodium cyanide (5.60 g, 114mmol) was added to the resulting solution. The reaction mixture washeated to 70° C. being stirred under nitrogen until the solution hadbecome almost colourless (overnight). This final solution was taken todryness under vacuum, with the residue extracted with boilingacetonitrile (3×25 mL). The total extract was filtered, reduced undervacuum to a white solid, and cooled to −10° C. to precipitate whitecrystals of the product. Drying in vacuo provided 3 (1.72 g, 58.5%). mp91˜94.0° C. ¹H-NMR (D₂O): δ 2.51 (s, 12H, NCCH₂N); 2.42 (s, 12H,NCH₂CH₂N). MS Calcd for C₁₄H₃₅N₈ [M+1-5H₂O]⁺ m/z 315.3, found 315.8.Elemental analysis calculated for C₁₄H₄₄N₈O₅, requires C, 41.56; H,10.96; N, 27.70, Found: C, 41.53; H, 10.28; N, 27.12.

Synthesis of [Cu(DiAmSar)]Cl₄.5H₂O (4)

CuCl₂.2H₂O (0.17 g, 1.0 mmol), was dissolved in 10 mL water, followingadded 3 (0.41 g, 1.0 mmol). The solution was acidified to pH=4.0 withHCl (0.1 M), and stirred overnight. Evaporation on heating yielded ablue precipitate. The blue solid was cooled, filtered, and washed withethanol (5 mL×3), and dried, yielding a blue crystalline product 4 (0.53g, 97%). ¹H-NMR (D₂O): δ 3.20 (s, 12H, NCH₂CH₂N); 2.90 (s, 12H, NCCH₂N).MS Calcd for C₁₄H₃₃CuN₈ [M+1-4HCl-5H₂O]⁺ m/z 376.2, found 376.0.Elemental analysis calculated for C₁₄H₄₆Cl₄CuN₈O₅, requires C, 27.48; H,7.58; N, 18.31; Cl, 23.17, Cu, 10.38, Found: C, 27.72; H, 7.17; N,17.87; Cl, 22.40, Cu, 9.71.

Synthesis of [Cu(AmMBSar)](CH₃COO)₂.5H₂O (5)

[Cu(DiAmSar)]Cl₄.5H₂O (0.73 g, 1.2 mmol) was dissolved in dry ethanol(30 mL), followed by addition of methyl 4-formylbenzoate (0.28 g, 1.7mmol), dried/activated 4 Å molecular sieves (1.0 g) and glacial aceticacid (60 μL). The resulting solution was stirred for 3 h under argongas, followed by addition of sodium cyanoborohydride (0.82 g, 14 mmol).The reaction mixture continued to stir under argon gas for 4 days atroom temperature 20˜25° C. The mixture was filtered, and filtrate wasevaporated to dryness and extracted with ethyl acetate (15 mL×3), driedand then diluted to 300 mL. It was placed onto a SP Sephadex C25 columnand eluted with sodium citrate (0.1 M, 400 mL) and a wide blue bandformed. Increasing the sodium citrate concentration (0.3 M, 500 mL)resulted in three blue bands eluting in order as [Cu(diamsar)]²⁺,[Cu(AmMBSar)]²⁺ (compound 5), and [Cu(DiAMBSar)]²⁺ by TLC monitoring.The second band eluate (compound 5 solution, 100 mL) was isolated anddiluted with water (10 fold, 1.0 L), and placed onto another SP SephadexC25 column. A single blue band eluted with sodium acetate (1.0 M, 200mL), was evaporated to dryness and the residue extracted with 2-propanol(50 mL×3). Fine white crystals of sodium acetate were separated,filtered and the process of evaporation and extraction repeated 3 times.The final residue was dried in vacuo to a dark blue solid 5[Cu(AmMBSar)](Ac)₂.5H₂O (246.5 mg, 27.9%). ¹H-NMR (D₂O): δ 8.0-7.9 (d,2H, aromatic); 7.48-7.42 (d, 2H, aromatic); 4.70 (s, 3H, OCH₃); 4.02 (s,2H, CH₂—Ar); 3.54-3.30 (m, 12H, NCH₂CH₂N); 2.93-2.78 (m, 12H, NCCH₂N).MS Calcd for C₂₃H₄₃CuN₈O₂[M+1-2HAc-5H₂O]⁺ m/z 526.3, found 526.8.

Synthesis of AmMBSar (6)

Sodium borohydride (150 mg) was dissolved in 0.4 mL water and stirredunder a nitrogen atmosphere, following addition of Pd/C (60 mg) in 1.0mL water. Compound 5 (164 mg) was dissolved in sodium hydroxide (3 mL;1% NaOH) and added dropwise to the above mixture solution. Stirring wascontinued under nitrogen at 25° C. until the color turned from blue toclear. The resulting solution was filtered (0.22 μm) and the filtratecollected in an ice-cooled glass vial. Concentrated hydrochloric acidwas added dropwise (50 μL) to the cooled solution until gas evolutionceased (˜450 μL, HCl). The solution was acidified to pH 4˜6, and thendried under vacuum to provide AmMBSar, 6 (56 mg, 47.6%). ¹H-NMR (D₂O): δ7.97-7.92 (d, 2H, aromatic); 7.50-7.45 (d, 2H, aromatic); 4.65 (s, 3H,OCH₃); 4.08 (s, 2H, CH₂—Ar); 3.45-3.20 (m, 12H, NCH₂CH₂N); 3.12-2.98 (m,12H, NCCH₂N); 1.98-1.85 (m, 9H, NH). MS Calcd for C₂₃H₄₂N₈O₂ [M+1]⁺ m/z463.0, found 462.3.

Synthesis of AmBaSar (7)

Compound 6 (46.2 mg, 0.1 mmol) was dissolved in methanol (3 mL) andwater (1.0 mL), followed by addition of NaOH (1.5 mL, 0.1 M). Theresulting solution was refluxed and stirred for 5 h, then neutralizedwith HCl (1.0 M) to pH 6˜7. Drying the solution under reduced pressureformed solids, which were dissolved with hot MeOH (3×2 mL). The totalextract was filtered and dried under vacuum to yield AmBaSar, 7 (36 mg,80.2%). ¹H-NMR (D₂O): δ 7.76-7.67 (m, 2H, aromatic); 7.45-7.37 (m, 2H,aromatic; 3.66 (s, 2H, NCH₂C) 3.26-3.04 (m, 12H, NCCH₂N); 2.99-2.84 (m,12H, NCH₂CH₂N); 1.84-1.77 (m, 9H, NH). MS Calcd for C₂₃H₄₁N₈O₂ [M+1]⁺m/z 449.3, found 449.7. A typical ¹H-NMR spectra of AmBaSar in D2O isshown in FIG. 3. A Typical HPLC chromatogram of AmBaSar using theAnalytical HPLC system is shown in FIG. 4.

Synthesis of Other Carboxylic Acid Functionalized Sar Based BifunctionalChelators

Although the methodology for functionalizing the apical amine has beendescribed primarily with respect to AmBaSar, Sar may be functionalizedwith other carboxylic acids by reacting with the compounds that have analdehyde on one side and the Methoxyl ester on the other. These twogroups could located at different positions of an aromatic system(including those with hetero-atoms), aliphatic system (including thosewith double or triple bonds), or the combination of those two.

Synthesis of Carboxylate and Thiol Functionalized Sar Based BifunctionalChelators

Using methods analogous to the preparation of AmBaSar, the carboxylateand thiol functionalized Sar cage is synthesized by a method shown inFIG. 21. Starting from the mono-substituted Sar cage and commerciallyavailable agents, a bifunctional linker with protected thiol andcarboxylate groups is obtained. After the removal of the Co metal anddeprotection, the carboxylate and thiol functionalized Sar cage isobtained. Various linkers may also be introduced to this Sar cage agent.

Synthesis of DiBaSar and Functionalized Sar Based Bifunctional ChelatorsHaving Carboxylic Acid Groups at Both Apical Amines

DiBaSar and Sar based structures functionalized with carboxylic acids atboth apical amines may be synthesized by a synthetic procedure describedin FIG. 22

Experimental: AmBaSar Conjugating Peptide RGD and RadiolabelingSynthesis of AmBaSar-RGD (8, FIG. 2)

AmBaSar was activated by EDC at pH 5.5 for 30 min (4° C.), with a molarratio of AmBaSar:EDC:SNHS=5:5:4. Typically, 15.0 mg of AmBaSar (30 μmol)was dissolved in 500 μL of water. Separately, 5.76 mg of EDC (30 μmol)was dissolved in 500 μL of water. The two solutions were mixed, and 0.1M NaOH (250 μL) was added to adjust the pH to 4.5. SNHS (5.2 mg, 24μmol) was then added to the stirring mixture on an ice-bath, followed by0.1 M NaOH (50 μL) to finalize the pH to 5.4. The reaction was allowedto stir for 30 min at 4° C. The theoretical concentration of activeester AmBaSar-OSSu was calculated to be 24 μmol. Cyclic RGD peptide (2.5mg, 4.0 μmol) dissolved in 500 μL (5.0 mg/mL) of water was cooled to 4°C. and added to the AmBaSar-OSSu reaction mixture. The pH was adjustedto 8.6 with 0.1 M NaOH (280 μL). The reaction was allowed to proceedovernight at room temperature (20˜25° C.). The AmBaSar-RGD conjugate waspurified by semipreparative HPLC. A representative chromatogram of theAmBaSar-RGD is shown in FIG. 5. The peak containing the RGD conjugatewas collected, lyophilized, and dissolved in water at a concentration of1.0 mg/mL for use in radiolabeling reactions. A mass spectra ofAmBaSar-RGD is shown in FIG. 6.

Synthesis of [Cu-AmBaSar-RGD](Ac)₂.2HAc

The AmBaSar-RGD (1.0 mg) was dissolved in 0.5 mL of a 0.1 M ammoniumacetate/0.80 mM copper (II) acetate solution. The mixture was stirred at37° C. for 40 min and allowed to cool to room temperature. The crudeCu-AmBaSar-RGD solution was purified and quantified by HPLC. MS Calcdfor C₅₈H₉₄N₁₆O₁₇Cu [M+1]⁺ m/z 1352.0, found 1351.9.

Radiolabeling of ⁶⁴Cu-AmBaSar-RGD

[⁶⁴Cu]Acetate (⁶⁴Cu(OAc)₂) was prepared by adding 111 MBq (3 mCi) of⁶⁴CuCl₂ in 0.1 M HCl into an 1.5 mL microfuge tube containing 300 μL 0.1M ammonium acetate (pH 5.0), followed by mixing using a mini vortex andincubating for 15 min at room temperature. The AmBaSar-RGD (1-2 μg in100 μL 0.1 M ammonium acetate) was labeled with ⁶⁴Cu(OAc)₂ by additionof 1-3 mCi of ⁶⁴Cu. The chelation reaction was performed in 0.1 M sodiumacetate buffer, pH 5.0, for 60 min at room temperature (23˜25° C.).Labeling efficiency was determined by HPLC. ⁶⁴Cu-AmBaSar-RGD waspurified by radio-HPLC. The elute was evaporated and the activityreconstituted in saline, followed by passage through a 0.22 μm Milliporefilter into a sterile dose vial for use in animal experiments. Arepresentative radio-HPLC chromatogram of ⁶⁴Cu-AmBaSar-RGD is shown inFIG. 7.

Improved Synthesis of AmBaSar

Another embodiment of the present invention is directed to an improvedsynthetic route of AmBaSar. The improved synthesis of the BFC AmBaSar isshown in FIG. 8. In this new synthetic route, AmBaSar is obtained inonly four steps starting from Co(DiAmSar)]Cl₅ (1). As shown in FIG. 8,Compound 1 was transformed to the 3Cl⁻ salt by neutralizing theprotonated primary amino groups with NaOH, which was then converted tothe tetraphenylborate salt compound 2 by anion exchange of[Co(DiamSar)]Cl₃ with sodium tetraphenylborate (NaBPh₄) in aqueoussolution. (Ref. 24) The coupling of the aldehyde to the amine ofcompound 2 yielded aromatic functionalization of the cage compound 3Co(AmBMSar) complex. This reaction was carried out under dehydrating andrefluxing conditions before adding the reducing agent according toliterature method. (Ref. 24) Molecular sieves 4 Å were employed toremove the water produced in the formation of the iminium ion. Comparedwith the Cu complex (27.9% yield), we also observed an increased yield(39.1% yield) in this step, which could be partially attributed to thepreferable solubility of the tetraphenylborate salt compared with thechloride salt in methanol. We also found that AmBaSar could be made intwo ways from compound 3. One method was similar to that discussedherein, where the Co(III) ion of compound 3 was removed by excesscyanide ion to yield the free cage compound 5 AmBMSar, which then washydrolyzed to produce the target compound 6 AmBaSar. Another methodemployed alkaline hydrolysis of compound 3 first, with subsequentremoval of cobalt ion to yield the AmBaSar (6).

This improved synthesis of AmBaSar shortens synthetic steps andsimplifies purification. The improved synthesis also simplified theseparation process and increased the yield. The overall yield wasincreased to about 6.0±0.2% (n=6) compared with the original 4.7% byusing tris(ethylenediamine)cobalt(III) chloride dehydrate as startingmaterial.

Materials.

All reagents and solvents were purchased from Sigma-Aldrich Chemicals(St. Louis, Mo., USA) and used without further purification unlessotherwise stated. DOTA was purchased from Macrocyclics (Dallas, Tex.,USA). ⁶⁴CuCl₂ was ordered from MDS Nordion (Vancouver, BC, Canada).Compound 1 (Co(DiAmSar)]Cl₅.H₂O) was prepared as described herein. Waterwas purified using a Milli-Q ultra-pure water system from MilliporeCorp. (Milford, Mass., USA).

General Methods:

¹H NMR spectra were obtained using a Varian Mercury 400 MHz instrument(USC NMR Instrumentation Facility), and the chemical shifts werereported in ppm on the δ scale relative to an internal TMS standard.Mass spectra using LC-MS were provided by the Proteomics Core Facilityof the USC School of Pharmacy. Thin-layer chromatography (TLC) wasperformed on silica gel 60 F-254 plates (Sigma-Aldrich) using a mixturesolution of 70% MeOH and 30% aqueous NH₄OAc (NH₄OAc solution is 20% byweight) as the mobile phase. Ion-exchange chromatography was performedunder gravity flow using Dowex 50WX2 (H⁺ form, 200-400 mesh) cationexchange resins. All evaporations were performed at reduced pressure(ca. 20 Torr) using a Büchi rotary evaporator.

HPLC was accomplished on two Waters 515 HPLC pumps, a Waters 2487absorbance UV detector and a Ludlum Model 2200 radioactivity detector,operated by Waters Empower 2 software. The analytical HPLC was performedon a Phenomenex Luna C18 reversed phase column (5 μm, 250×4.6 mm) andmonitored using a radiodetector and UV at 254 nm. The flow was 1.0mL/min, with the mobile phase starting from 99% solvent B (0.1% TFA inwater) and 1% solvent C (0.1% TFA in acetonitrile) (0˜1 min) to 70%solvent B (0.1% TFA in water) and 30% solvent C (0.1% TFA inacetonitrile) (1˜30 min). Radio-TLC was performed with MKC18 silica gel60 Å plates (Whatman, N.J.) with solvent MeOH/20% NaOAc=3:1 as theeluent using a Bioscan AR2000 imaging scanner (Washington, D.C.) andWinscan 2.2 software.

Synthesis of (1,8-Diamine-Sar) Cobalt (III) tri(tetraphenylborate)([Co(DiAmSar)](BPh₄)₃.H₂O, 2)

Compound 1 Co(DiAmSar)]Cl₅.H₂O (3.2 g, 5.5 mmol) was dissolved in water(20 mL), followed by the addition of 2 eq NaOH solution (11.0 mmol,sufficient to neutralize the protonated primary amino groups). Theresulting mixture was stirred for 30 min at room temperature until pHwas near to 7˜8. Sodium tetraphenylborate (5.2 g, 16.5 mmol) in 20 mLmethanol was added, and then stirred for 1 h. After filtration anddrying under reduced pressure, 6.6 g of compound 2[Co(DiAmSar)](BPh₄)₃.H₂O (85% yield) was obtained as an orange solid.MS: Calcd for C₈₆H₉₆B₃CoN₈O [M+1-2HBPh₄-H₂O]⁺ m/z 691.7,[M+1-3HBPh₄-H₂O]⁺ 371.4, found 692.1, 371.1. ¹H-NMR (DMSO-d6): 7.20-6.79(m, 45H, aromatic); 3.10-2.80 (m, 12H, NCCH₂N); 2.60-2.15 (m, 12H,NCH₂CH₂N).

Synthesis of (1-amine, 8-(aminomethyl) 4′-methylbenzoate-Sar) Cobalt(III) Pentachloride ([Co(AmBMSar)]Cl₅.5H₂O, 3)

Compound 2 [Co(DiAmSar)](BPh₄)₃.H₂O (7.6 g, 5.7 mmol) was dissolved inanhydrous MeOH (40 mL) and methyl 4-formylbenzoate (1.6 g, 10.0 mmol),followed by addition of 1.7 mL acetic acid and 4 Å molecular sieves. Thesolution was refluxed 72 h under argon and shielded from visible light.The reaction mixture was cooled to room temperature, and sodiumtriacetoxyborohydride (NaHB(OAc)₃, 9.7 g, 45.0 mmol) was added. Theresulting mixture was stirred overnight. The products were loaded ontosilica gel and initially purified by flash column chromatography, usinga mixture of 70% MeOH and 30% aqueous NH₄OAc solution as the elutingsolvent. The fractions were monitored by silica gel TLC. This initialcolumn chromatography partially separated the biscoupled Co-cage complexand the mono-coupled Co-cage [Co(AmBMSar)] complex from the startingmaterial. Each product was further purified by evaporating the MeOH,diluting the remaining aqueous solution ˜10-fold, and loaded on a Dowex50-WX2 [H⁺] cation exchange resin column. The [Co(AmBMSar)] complex waseluted from the column with 3.0-4.0 M HCl. The HCl eluant was evaporatedunder reduced pressure to give a yellow solid compound 3[Co(AmBMSar)]Cl₅.5H₂O (1.8 g, 39.1% yield). MS: Calcd forC₂₃H₅₄Cl₅CoN₈O₇ [M+Na-2HCl-5H₂O]⁺ m/z 650.2, [M-5HCl-5H₂O+Na]⁺ 541.2,found 649.7, 541.2. ¹H-NMR (D₂O): 7.93-7.86 (m, 2H, aromatic); 7.42-7.35(m, 2H, aromatic); 4.05 (s, 3H, CCH₃); 3.75 (s, 2H, NCH₂C); 3.45-3.26(m, 12H, NCCH₂N); 2.88-2.75 (m, 12H, NCH₂CH₂N).

Synthesis of (1-amine, 8-(aminomethyl) 4′-carboxybenzene-Sar) Cobalt(III) Trichloride ([Co(AmBaSar)]Cl₃.5H₂O, 4)

Compound 3 [Co(AmSarBM)]Cl₅.5H₂O (2.4 g, 3.0 mmol) was dissolved in thesolution of methanol (40 mL)/water (30 mL) containing potassiumcarbonate (3.0 g), and refluxed for 5 h. The reaction mixture was thenneutralized with hydrochloric acid, diluted to 1 L, sorbed on Dowex50-WX2 [H⁺] cation exchange resin column, and eluted with hydrochloricacid (2˜3 M, 2 L). The dark red fraction was collected and evaporated todryness. 1.8 g compound 4 was obtained in 89% yield. MS: Calcd forC₂₂H₅₀Cl₃CoN₈O₇ [M+Na]+m/z 724.2, [M+Na-5H₂O]⁺ 634.2, found 724.1,634.3. ¹H-NMR (D₂O): 7.91-7.87 (m, 2H, aromatic); 7.42-7.36 (m, 2H,aromatic); 4.00 (s, 2H, NCH₂C); 3.47-3.27 (m, 12H, NCCH₂N); 3.07-2.73(m, 12H, NCH₂CH₂N).

Synthesis of. Methyl 4-((8-amino-3, 6, 10, 13, 16, 19-hexaazabicyclo[6.6.6] icosane-1-ylamino) methyl) benzoate (AmMBSar, 5)

Compound 3 [Co(AmBMSar)]Cl₅.5H₂O (790 mg, 1.0 mmol), NaOH (95 mg,sufficient to neutralize the protonated primary amino groups) andCoCl₂.6H₂O (260 mg) were dissolved in deoxygenated water (20 mL) undernitrogen. The solution turned green from brown after NaCN (1.0 g) wasadded. The resulting mixture continued to react in 70° C. under nitrogenuntil the solution had become almost colourless (overnight). This finalsolution was evaporated under reduced pressure, and the residue wasextracted with boiling acetonitrile (3×15 mL). The total extract wasfiltered, and dried under vacuum to provided compound 5 AmMBSar (110 mg,24.5% yield).

Synthesis of AmBaSar (6)

AmBaSar was prepared by two methods:

Method 1. This is similar to the preparation of AmMBSar from compound 3.Briefly, the cobalt ion on compound 4 was removed to produce the targetcompound 6 AmBaSar (26% yield) by reduction with excess cyanide ion toyield the free cage.

Method 2. Compound 5 was alkaline hydrolyzed to produce the targetcompound 6 AmBaSar (80.2%, yield).

Radiolabeling of BFC AmBaSar and DOTA, Evaluation of RadiolabeledAmBaSar and Comparison with Radiolabeled DOTA.

FIG. 9 shows a method for the ⁶⁴Cu radiolabeling of AmBaSar according tothe present invention. AmBaSar (2.5˜25 μg) was labeled with ⁶⁴Cu (1˜5mCi) in 0.1 M ammonium acetate solution (pH 5.0) at room temperature(23˜25° C.) for 5 min to 30 min. After 5 min complexation, theradiolabeling yield of ⁶⁴Cu-AmBaSar was more than 97%. The AmBaSar wasnearly quantitatively labeled with ⁶⁴Cu²⁺ within 30 min under the aboveexperimental conditions. The complexation rates by the AmBaSar at pH 5with ⁶⁴Cu²⁺ was satisfactory for its use in the development of⁶⁴Cu-radiopharmaceuticals. In conclusion, the new functionalized AmBaSarcan efficiently label ⁶⁴Cu²⁺ at room temperature due to the provision ofa three-dimensional hexa-aza cage by increasing thermodynamic andkinetic stability to the ⁶⁴Cu²⁺ complex, which is similar with other Sarbased ligands.

The radiochemical yield of ⁶⁴Cu-AmBaSar was determined by radio-HPLC orradio-TLC after different time points. In HPLC, the free ⁶⁴Cu was elutedat about 3.6 min, while the retention time of ⁶⁴Cu-AmBaSar was 17.9 min.A typical HPLC of crude ⁶⁴Cu-AmBaSar is shown in FIG. 10. For TLC, theR_(f) value of free ⁶⁴Cu and complexed ⁶⁴Cu-AmBaSar were well-separated.⁶⁴Cu remained at the origin (R_(f)=0) and the ⁶⁴Cu-AmBaSar complex closeto solvent front (R_(f)=0.71). The radiochemical yield of ⁶⁴Cu-AmBaSarwas determined to be more than 98%.

⁶⁴Cu-DOTA was prepared following literature procedures. (Refs. 25, 26)The radiochemical yield of ⁶⁴Cu-DOTA was determined by radio-TLC afterdifferent time points. The radiochemical yield of ⁶⁴Cu-DOTA was morethan 98% after the complexation.

Lipophilicity (Octanol/Water Partition Coefficient) Studies.

Information about the lipophilicity of the ⁶⁴Cu-AmBaSar and ⁶⁴Cu-DOTAwas obtained by measuring their partitioning in a 1-octanol/watersystem. Counts in samples were used to determine partition coefficients(log P) values, calculated using a known formula. The log P values weremeasured at pH 7.4 in PBS. The log P value of ⁶⁴Cu-AmBaSar is −2.6, andthe corresponding value of ⁶⁴Cu-DOTA is −2.3. Both ⁶⁴Cu-AmBaSar and⁶⁴Cu-DOTA are hydrophilic. This indicates that they should bepredestined to show rapid blood clearance and preferential renalexcretion.

In Vitro Studies.

The stability of the BFC copper-64 complex under physiologicalconditions is very important. In vitro stability of ⁶⁴Cu-AmBaSar in PBS(pH 7.4) and FBS solutions at physiological temperatures was tested andthe results are shown in FIG. 11. In vitro stability of ⁶⁴Cu-AmBaSar wasdetermined in the PBS or FBS using HPLC or TLC after incubating fordifferent time intervals (2, 4, 20, and 26 h). The radiochemical purityof ⁶⁴Cu-AmBaSar in the PBS or FBS is more than 97% after 26 hoursincubation. The stability of ⁶⁴Cu-AmBaSar in mouse blood was also. Over98% of ⁶⁴Cu-AmBaSar remained untouched after its incubation for 4 h.These in vitro stability studies demonstrated that the ⁶⁴Cu-AmBaSar isvery stable in PBS, FBS, and the blood at physiological pH.

In Vivo Studies.

MicroPET imaging and biodistribution studies were performed with thenon-targeted ⁶⁴Cu-AmBaSar and ⁶⁴Cu-DOTA complexes to allow a comparisonof their base-line organ uptake and clearance properties. The micro-PETimaging results were shown in FIG. 12. After 30 min, the radioactivityquickly cleared from muscle and blood, and mainly localized in thebladder and kidneys. Regarding ⁶⁴Cu-DOTA, the radioactivity mainlylocalized in the bladder, kidneys, and liver. At the 30 min time point,both ⁶⁴Cu-DOTA and ⁶⁴Cu-AmBaSar showed high kidney uptake, with⁶⁴Cu-DOTA having a much higher liver uptake than ⁶⁴Cu-AmBaSar. By twohours renal activity cleared significantly for both agents, althoughliver and bowel activity remained in the animals receiving ⁶⁴Cu-DOTA.Although more detailed pharmacokinetic analyses are required, renalactivity for ⁶⁴Cu-AmBaSar decreased by 51% between 30 min and 2 hoursbased on the micro-PET region of interest quantification, comparingfavorably with ⁶⁴Cu-DOTA clearance (66%).

The biodistribution of ⁶⁴Cu-AmBaSar and ⁶⁴Cu-DOTA were assessed inBalb/c mice. Each animal was sacrificed at the time interval 2 h and theorgans dissected. The values of percent injected dose/gram (ID/g %) indifferent organs were compared and illustrated in FIG. 13. The⁶⁴Cu-AmBaSar cleared rapidly from the blood and predominantly throughthe kidneys. After 2 h, the kidney and liver uptake reached 1.09±0.27,0.14±0.01% ID/g, respectively, which is consistent with the excretionpattern from the microPET imaging results. The ⁶⁴Cu-DOTA cleared throughthe kidney and liver, with uptake reached 1.28±0.41, 1.25±0.09,respectively, by 2 h.

Copper ion in vivo is responsible for many enzymatic processes and hencecan be bound by many proteins. Its natural excretory path is via thehepatocytes in the liver. It may be recirculated by binding toceruloplasmin in the liver. While rapid clearance through the kidneys istypical of charged ⁶⁴Cu-complexes which maintain their identity,hepatobiliary clearance of ⁶⁴Cu-radiopharmaceuticals is much lessdesirable, and its presence usually suggests loss of Cu-64 from theligand. (1, 28) Prasanphanich A F, Retzloff L, Lane S R, Nanda P K,Sieckman G L, Rold T L, et al. In vitro and in vivo analysis of[⁶⁴Cu—NO2A-8-Aoc-BBN(7-14)NH₂]: a site-directed radiopharmaceutical forpositron-emission tomography imaging of T-47D human breast cancertumors. Nucl Med Biol 2009; 36:171-81. Rapid renal clearance, as opposedto mixed renal and hepatic clearance resulting from administration of⁶⁴Cu-DOTA, will provide better somatic contrast from the imagingperspective, and potentially reduce radiation exposure for therapeuticanalogues. Therefore, the development of more stable chelators would bebeneficial as free Cu clears through liver. The hydrophilicity of⁶⁴Cu-AmBaSar (log P=−2.6) is only slightly different from ⁶⁴Cu-DOTA (logP=−2.3). Both compounds should mainly clear through the kidneys. For⁶⁴Cu-AmBaSar, residual kidney uptake and low liver accumulation at 2 hpost injection were observed in both the microPET images andbiodistribution studies, compared with high residual liver and bowelaccumulation with ⁶⁴Cu-DOTA. However, with similar hydrophilicity, theliver uptake of ⁶⁴Cu-DOTA is eight times higher than that of⁶⁴Cu-AmBaSar. The comparative data from microPET imaging andbiodistribution studies between ⁶⁴Cu-AmBaSar and ⁶⁴Cu-DOTA supports theassumption that ⁶⁴Cu-AmBaSar is more stable in vivo than ⁶⁴Cu-DOTA.Nonetheless, the differential organ uptake and clearance propertiescould also be partially attributed to the characteristics of the BFCAmBaSar with polyamine functional groups, and DOTA with polyacidfunctional groups. Although accumulation of radioactivity in liver islikely related to the loss ⁶⁴Cu from the DOTA chelator, furtherbiochemical testing may be required to determine the disposition of ⁶⁴Cuin liver.

Materials and Methods

Bifunctional Chelator AmBaSar Labeling Copper-64.

[⁶⁴Cu]Acetate (⁶⁴Cu(OAc)₂) was prepared by adding 185 MBq (5 mCi) of⁶⁴CuCl₂ in 0.1 M HCl into an 1.5 mL microfuge tube containing 300 μL 0.1M ammonium acetate (pH 5.0). The mixture was vortexed and incubated for15 min at room temperature. AmBaSar (2.5-25 μg), diluted in 100 μL of0.1 M ammonium acetate (pH 5.0), was mixed with 1˜5 mCi of ⁶⁴Cu(OAc)₂.The solution was then incubated at room temperature (23˜25° C.) for 5min˜30 min. The radiochemical yield was determined by radio-HPLC orradio-TLC at different time points. ⁶⁴Cu-AmBaSar was purified byradio-HPLC, and the eluant was evaporated and reconstituted in saline,which was filtered into a sterile dose vial for use in animalexperiments by passage through a 0.22 μm Millipore filter.

Bifunctional Chelator DOTA Labeling Copper-64.

DOTA (20 μg) was labeled with ⁶⁴Cu²⁺ by addition of 2 mCi of ⁶⁴Cu²⁺ in0.1 M sodium acetate buffer (pH 5.5) followed by 45 min incubation at45° C. The radiochemical yield was determined by radio-TLC (the samecondition with ⁶⁴Cu-AmBaSar).

Determination of the Partition Coefficient Log P.

The partition coefficient value was expressed as log P. Log P of⁶⁴Cu-AmBaSar or ⁶⁴Cu-DOTA was determined by measuring the distributionof radioactivity in 1-octanol and PBS. A 5 μL sample of ⁶⁴Cu-AmBaSar or⁶⁴Cu-DOTA in PBS was added to a vial containing 1 mL each of 1-octanoland PBS. After vortexing for 5 min, the vial was centrifuged for 5 minto ensure complete separation of layers. Then, 5 μL of each layer waspipetted into other test tubes and radioactivity was measured using agamma counter (Packard). The measurement was repeated three times.

In Vitro Stability Assessment.

The stability of the ⁶⁴Cu-AmBaSar was assayed by measuring theradiochemical purity after different incubation times at roomtemperature or 37° C. For the in vitro stability study, 100 μCi of the⁶⁴Cu-AmBaSar was pipetted into 1 mL of PBS and fetal bovine serum (FBS),respectively, followed by incubation in PBS at room temperature and inFBS at 37° C. An aliquot of the mixture was removed for thedetermination of radiochemical purity by HPLC at different time pointsafter incubation (2, 4, 20, and 26 h). The solution of FBS wascentrifugated, and the upper solution was taken and filtered for HPLCanalysis.

MicroPET Imaging and Biodistribution.

MicroPET imaging and biodistribution of the radiolabeled BFCs wereperformed on Balb/c mice to evaluate their in vivo properties. Animals(Balb/c, n=2) were injected with 0.3 mCi ⁶⁴Cu-AmBaSar or ⁶⁴Cu-DOTAthrough the tail vein. PET imaging (10-min static scans) was performedusing a microPET R4 scanner (Concorde Microsystems, Inc, Knoxville,Tenn.), with 120 transaxial planes and spatial resolution of 1.2 mm, at30 min and 2 h postinjection. The microPET data were reconstructed usingthe ordered subsets expectation maximization (OSEM) algorithm using themicroPET Manager Software (CTI Concorde Microsystems, Inc, Knoxville,Tenn.). Images were then analyzed using the Acquisition Sinogram ImageProcessing (ASIPro) software (CTI Concorde Microsystems, Inc, Knoxville,Tenn.).

For biodistribution studies, one group of mice (Balb/c, n=3) wasinjected intravenously with the radiotracer ⁶⁴Cu-AmBaSar (10 μCi, 100μL). Another group of mice was injected similarly with ⁶⁴Cu-DOTA (10μCi, 100 μL). Activity injected into each mouse was measured in a dosecalibrator (Capintec). After inhalation anesthesia, animals weresacrificed at 2 h postinjection. Tissues and organs of interest wereseparated and weighed. Radioactivity in each organ was measured using agamma counter, and radioactivity uptake was expressed as % ID/g. Meanuptake (% ID/g) for each group of animals was calculated with standarddeviations.

Copper-64 Labeled AmBaSar Conjugated Cyclic RGD Peptide for ImprovedMicroPET Imaging of Integrin α_(v)β₃ Expression

⁶⁴Cu-AmBaSar-RGD was obtained in high yield under mild conditions forPET imaging of integrin α_(v)β₃ expression. This tracer exhibits goodtumor-targeting efficacy, excellent metabolic stability, as well asfavorable in vivo pharmacokinetics. In vitro and in vivo evaluation ofthe ⁶⁴Cu-AmBaSar-RGD shows it has improved in vivo stability comparedwith the established tracer ⁶⁴Cu-DOTA-RGD. The AmBaSar chelator hasgeneral application for ⁶⁴Cu labeling of various bioactive molecules inhigh radiochemical yield and high in vivo stability for future PETapplications.

Syntheses of AmBaSar-RGD and DOTA-RGD

AmBaSar could be activated and conjugated to the cyclic RGDyk peptide ina water-soluble procedure as described herein. The conjugation can alsobe performed in organic phase according to literature procedures (Refs.29, 30) In brief: the solution of AmBaSar (4.5 mg, 0.01 mmol), HATU (3.8mg, 0.01 mmol), HOAt (1.4 mg, 0.01 mmol), and dimethyl sulfoxide (DMSO,0.5 mL) were stirred at room temperature for 10 min. Seven equivalent ofDIPEA (9.1 mg, 0.07 mmol) and cyclic RGDyk (1.2 mg, 0.002 mmol) in 300μL DMSO were then added to the mixture at 0° C. The mixture was stirredfor 3 h at room temperature, and the solvent removed in vacuo. Theresidue was dissolved in acetonitrile/water (1:3) containing 0.1% TFAand purified by semipreparative HPLC. AmBaSar-RGD was obtained as awhite solid material after lyophilization. DOTA was activated andconjugated to cyclic RGDyk according to literature methods. (Ref. 31)

DOTA-RGD was also purified by semipreparative HPLC and confirmed by massspectrometry. AmBaSar-RGD and DOTA-RGD conjugates were dissolved in 0.1N ammonium acetate buffer solution (pH 5˜5.5), respectively, and storedin −20° C. for the future use in radiolabeling reactions.

Copper-64 Labeling and Formulation.

The ⁶⁴Cu-AmBaSar-RGD was prepared as described supra with minormodifications as follows: [⁶⁴Cu]Acetate (⁶⁴Cu(OAc)₂) was prepared byadding 37-111 MBq of ⁶⁴CuCl₂ in 0.1 N HCl into 300 μL 0.1 N ammoniumacetate buffer (pH 5.0˜5.5), followed by mixing and incubating for 15min at room temperature. The AmBaSar-RGD (about 2˜5 μg in 100 μL 0.1 Nammonium acetate buffer) was added to the above ⁶⁴Cu(OAc)₂ solution. Theresulting mixture was incubated at temperature 23˜37° C. for 30 min. The⁶⁴Cu-AmBaSar-RGD was determined and purified by semipreparative HPLC.The radioactive peak containing ⁶⁴Cu-AmBaSar-RGD was collected andconcentrated by rotary evaporation to remove organic solvent. And theradioactivity was reconstituted in 500-800 μL phosphate buffered saline(PBS), and passed through a 0.22 μm Millipore filter into a sterile dosevial for use in experiments below.

Details of the ⁶⁴Cu-labeling DOTA-RGD procedure were reported earlier.(Ref. 31) In brief, 37˜111 MBq of ⁶⁴CuCl₂ in 0.1N HCl were diluted in300 μL of 0.1 N ammonium acetate buffer (pH 5.5), and added to theDOTA-RGD solution (about 2˜5 μg in the 100 μL 0.1 N ammonium acetatebuffer). The reaction mixture was incubated at 45° C. for 45 min. Theradiochemical yield was determined by radio-TLC and HPLC. ⁶⁴Cu-DOTA-RGDwas then purified by semipreparative HPLC, and the radioactive peakcontaining the desired product was collected. After removal of thesolvent by rotary evaporation, the residue was reconstituted in 500-800μL of PBS and passed through 0.22 μm Millipore filter into a sterilemultidose vial for use in following experiments.

Determination of Log P Value.

The partition coefficient value was expressed as log P. Log P of⁶⁴Cu-AmBaSar-RGD was determined by measuring the distribution ofradioactivity in 1-octanol and PBS. A 5 μL sample of ⁶⁴Cu-AmBaSar-RGD inPBS was added to a vial containing 1 mL each of 1-octanol and PBS. Aftervigorously vortexing for 10 min, the vial was centrifuged for 5 min toensure the complete separation of layers. Then, 3×10 μL of each layerwas pipetted into other test tubes and radioactivity was measured usinga gamma counter (Perkin-Elmer Packard Cobra). The measurement wasrepeated three times, and Log P values were calculated according to aknown formula.

In Vitro Stability Assay.

The in vitro stability of the ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD werestudied at different time points. In brief, 3.7 MBq of the⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD were pipetted into 1 mL of the PBS,fetal bovine serum (FBS), and mouse serum, respectively. After incubatedat 37° C. for 3, 18, and 24 h, an aliquot of the mixture was removedfrom the PBS solution and the radiochemical purity was determined withHPLC. For the solution of FBS and mouse serum, the aliquots were addedto 100 μL PBS with 50% TFA. After centrifugation, the upper solution wastaken and filtered for HPLC analysis.

Cell Uptake Study.

U87MG human glioblastoma cell line (integrin α_(v)β₃-positive) wasobtained from the American Type Culture Collection (ATCC, Manassas, Va.)and maintained under standard conditions according to ATCC as following:The U87MG glioma cells were grown in Gibco's Dulbecco's mediumsupplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin,and 100 μg/mL streptomycin (Invitrogen Co, Carlsbad, Calif.), at 37° C.in humidified atmosphere containing 5% CO₂. The U87MG glioma cells weregrown in culture until sufficient cells were available.

The cell uptake study was performed as described in the literature withsome modifications. (Ref. 30) Cells were incubated with ⁶⁴Cu-AmBaSar-RGD(37 kBq/well) or ⁶⁴Cu-DOTA-RGD at 37° C. for 30 min, 60 min, and 120min. The blocking experiment was performed by incubating U87MG cellswith ⁶⁴Cu-AmBaSar-RGD (37 kBq/well) in the presence of 2 μg RGD. Thetumor cells were then washed three times with chilled PBS and harvestedby 0.1 N NaOH solution containing 0.5% sodium dodecyl sulfate (SDS). Thecell suspensions were collected and measured in the gamma counter.

Animal Model.

Athymic nude mice (about 10-20 weeks old, with a body weight of 25-35 g)were obtained from Harlan (Charles River, Mass.). All animal experimentswere performed according to a protocol approved by University ofSouthern California Institutional Animal Care and Use Committee. TheU87MG human glioma xenograft model was generated by subcutaneousinjection of 5×10⁶ U87MG human glioma cells into the front flank ofathymic nude mice. The tumors were allowed to grow 3-5 weeks until200-500 mm³ in volume. Tumor growth was followed by caliper measurementsof the perpendicular dimensions.

MicroPET Imaging and Blocking Experiment.

MicroPET scans and imaging analysis were performed using a rodentscanner (microPET R4; Siemens Medical Solutions) as previously reported(26). [26] Li, Z., Cai, W., Cao, Q., Chen, K., Wu, Z., He, L., and Chen,X. (2007) ⁶⁴Cu-labeled tetrameric and octameric RGD peptides forsmall-animal PET of tumor α_(v)β₃ integrin expression. J Nucl Med 48,1162-71. About 11.1 MBq of ⁶⁴Cu-AmBaSar-RGD or ⁶⁴Cu-DOTA-RGD wasintravenously injected into each mouse under isoflurane anesthesia. Tenminute static scans were acquired at 1, 2, 4, and 20 h post injection.The images were reconstructed by a 2-dimensional ordered-subsetsexpectation maximum (OSEM) algorithm. For each microPET scan, regions ofinterest were drawn over the tumor, normal tissue, and major organs onthe decay-corrected whole-body coronal images. The radioactivityconcentration (accumulation) within the tumor, muscle, liver, andkidneys were obtained from the mean value within the multiple regions ofinterest and then converted to % ID/g. For the receptor blockingexperiment, mice bearing U87MG tumors were scanned (10 min static) at 2h time point after the coinjection of 11.1 MBq of ⁶⁴Cu-AmBaSar-RGD or⁶⁴Cu-DOTA-RGD with 10 mg/kg c(RGDyK) per mouse.

Biodistribution Studies.

The U87MG tumor bearing nude mice (n=3) were injected with 0.37 MBq of⁶⁴Cu-AmBaSar-RGD or ⁶⁴Cu-DOTA-RGD to evaluate the biodistribution ofthese tracers. All mice were sacrificed and dissected at 20 h after theinjection of the tracers. Blood, U87MG tumor, major organs, and tissueswere collected and weighed wet. The radioactivity in the tissues wasmeasured using the gamma counter. The results were presented aspercentage injected dose per gram of tissue (% ID/g). For each mouse,the radioactivity of the tissue samples was calibrated against a knownaliquot of the injected activity. Mean uptake (% ID/g) for each group ofanimals was calculated with standard deviations.

Metabolic Stability.

The metabolic stabilities of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD wereevaluated in an athymic nude mouse bearing a U87MG tumor. Sixty minutesafter intravenous injection of 11.1 MBq of ⁶⁴Cu-AmBaSar-RGD or⁶⁴Cu-DOTA-RGD, the mouse was sacrificed and relevant organs wereharvested. The blood was collected and immediately centrifuged for 5 minat 13,200 rpm, and the upper serum solution was added to 100 μL PBSsolution containing 50% TFA, followed by mixing and centrifugation for10 min, and the upper solution was then taken out and filtered for HPLCanalysis. Liver, kidneys, and tumor were homogenized using ahomogenizer, suspended in 1 mL of PBS buffer, and centrifuged for 10 minat 14,000 rpm, respectively. For each sample, after removal of thesupernatant, the solution was added to 100 μL PBS solution containing50% TFA, followed by mixing and centrifugation for 10 min, and the uppersolution was then taken and filtered for HPLC analysis. Radioactivitywas monitored using a solid-state radiation detector. The eluent wasalso collected using a fraction collector (1.5 min/fraction) and theradioactivity of each fraction was measured with the γ counter.

Statistical Analysis.

Quantitative data were expressed as mean±SD. Means were compared usingOne-way ANOVA and student's t-test. P values <0.05 were consideredstatistically significant.

Results

Chemistry and Radiolabeling.

The AmBaSar-RGD conjugate was obtained in about 80% yield after HPLCpurification for both aqueous and organic phase procedures. The⁶⁴Cu-labeling (n=7) was achieved in more than 95% decay-corrected yieldfor ⁶⁴Cu-AmBaSar-RGD with radiochemical purity of >99%, and 80%radiochemical yield for ⁶⁴Cu-DOTA-RGD with radiochemical purity >98%.⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD were analyzed and purified by HPLC.The HPLC retention times of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD was 26.5min and 21.5 min, respectively, under the analytical condition. Forradio-TLC analysis, the free ⁶⁴Cu²⁺ remained at the origin of TLC plate,while the R_(f) values of ⁶⁴Cu-DOTA-RGD and ⁶⁴Cu-AmBaSar-RGD were about0.8˜1.0. The specific activity of ⁶⁴Cu-DOTA-RGD and ⁶⁴Cu-AmBaSar-RGD wasestimated to be about 10.1-22.2 GBq/μmol. Both tracers were usedimmediately after the formulation.

Log P Value and In Vitro Stability.

The octanol/water partition coefficients (log P) for ⁶⁴Cu-AmBaSar-RGDand ⁶⁴Cu-DOTA-RGD were −2.44±0.12 and −2.80±0.04, respectively (Ref.31), which demonstrated that both tracers are rather hydrophilic. The invitro stability of ⁶⁴Cu-AmBaSar-RGD was also studied in PBS (pH 7.4),FBS, and mouse serum for different time intervals (3, 18, and 24 h) atphysiological temperature 37° C. The stability was presented aspercentage of intact ⁶⁴Cu-AmBaSar-RGD based on the HPLC analysis and theresults are shown in FIG. 15. After 24 h incubation, more than 97% of⁶⁴Cu-AmBaSar-RGD remained intact in the PBS and FBS, and more than 95%of ⁶⁴Cu-AmBaSar-RGD remained intact in mouse serum. We also found that⁶⁴Cu-DOTA-RGD demonstrated similar stability results in vitro under theabove experimental conditions (data not shown).

In Vitro Cell Uptake.

Cell uptake study of ⁶⁴Cu-AmBaSar-RGD or ⁶⁴Cu-DOTA-RGD was performed onU87MG tumor cells, and the cell uptake was expressed as radioactivity(cpm) per 10⁶ cells after decay correction as shown in FIG. 16. The celluptake study revealed that both ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD couldbind to U87MG tumor cells, but relatively low amounts of activity (onlyabout 0.1˜0.4% was internalized). However, this binding could beefficiently blocked by excess amount of cold cyclic RGD peptide, whichdemonstrated the binding specificity of the radiolabeled ligands.

MicroPET Imaging.

The tumor-targeting efficacy and biodistribution patterns of⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD were evaluated in nude mice bearingU87MG human glioma xenograft tumors (n=3) at multiple time points (1, 2,4, and 20 h) with static microPET scans. Representative decay-correctedcoronal images at different time points were shown in FIG. 17A. TheU87MG tumors were clearly visualized with high tumor-to-backgroundcontrast for both tracers. The uptake of ⁶⁴Cu-AmBaSar-RGD in U87MGtumors was 2.04±0.14, 1.85±0.16, 1.87±0.11, and 0.97±0.05% ID/g at 1, 2,4, and 20 h p.i., respectively. ⁶⁴Cu-DOTA-RGD demonstrated similar tumoruptake with the value of 2.03±0.10, 1.97±0.05, 1.88±0.29, and 0.88±0.07%ID/g at the above time points, respectively (FIG. 4B-3). However, thebiodistribution patterns of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD weresignificantly different, especially in the kidneys and liver. The renaluptake of ⁶⁴Cu-AmBaSar-RGD were 2.83±0.77, 2.68±0.55, 2.49±0.50, and1.43±0.35% ID/g at 1, 2, 4, and 20 h p.i., respectively; while thecorresponding uptake for ⁶⁴Cu-DOTA-RGD was 1.52±0.46, 1.33±0.23,1.55±0.61, and 1.58±0.13% ID/g, respectively. This was lower than thatof ⁶⁴Cu-AmBaSar-RGD from 1 to 4 h, and reached comparable levels at 20 htime point. The liver uptake for ⁶⁴Cu-AmBaSar-RGD was 0.89±0.13,0.76±0.13, 0.75±0.14, and 0.64±0.06% ID/g at 1, 2, 4, and 20 h p.i. Thecorresponding uptake value for ⁶⁴Cu-DOTA-RGD was 2.28±1.08, 2.31±1.34,2.15±0.90, and 2.52±1.43% ID/g respectively, which were significantlyhigher than those of ⁶⁴Cu-AmBaSar-RGD at all time points.

Blocking Experiment.

The integrin α_(v)β₃ receptor specificity of ⁶⁴Cu-AmBaSar-RGD wasconfirmed by a blocking experiment where the tracers were co-injectedwith c(RGDyK) (10 mg/kg). As can be seen from FIG. 18, the U87MG tumoruptake in the presence of non-radiolabeled RGD peptide (0.09±0.03% ID/g)is significantly lower than that without RGD blocking (1.85±0.16% ID/g)(P<0.05) at 2 h p.i. The uptake of ⁶⁴Cu-AmBaSar-RGD in other organs(heart, intestine, kidneys, lungs, liver, and spleen) was alsosignificantly decreased, which correlates well with previous references.(Ref. 31) Similarly, the integrin α_(v)β₃ specificity of ⁶⁴Cu-DOTA-RGDwas confirmed by blocking experiments (FIG. 18).

Biodistribution Studies.

To validate the accuracy of small animal PET quantification, we alsoperformed a biodistribution experiment by using the direct tissuesampling technique. The data shown as the percentage administeredactivity (injected dose) per gram of tissue (% ID/g) in FIG. 19A. Both⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD mainly accumulated in kidneys, liver,stomach, intestine, and tumor. After 20 h p.i., the kidneys and liveruptake reached 1.51±0.27 and 0.55±0.06% ID/g for ⁶⁴Cu-AmBaSar-RGD, and0.98±0.40 and 2.16±0.85% ID/g for ⁶⁴Cu-DOTA-RGD, respectively. Thisdifference was consistent with the excretion pattern from the microPETimaging results. Based on the biodistribution results, we alsocalculated the contrast of tumor to main organs, which is shown in FIG.19B. For ⁶⁴Cu-AmBaSar-RGD, the ratio of tumor uptake to muscle, heart,lung, liver, and kidney uptake was 14.20±3.84, 7.33±1.55, 3.34±0.70,1.18±0.05, and 0.43±0.06, respectively; while the corresponding valuefor ⁶⁴Cu-DOTA-RGD were 5.97±2.25, 1.78±0.37, 1.27±0.37, 0.33±0.09, and0.66±0.04, respectively.

Metabolic Stability of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD.

The metabolic stability of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD weredetermined in mouse blood and in liver, kidney and tumor homogenates at1 h after intravenous injection of radiotracer into a U87MGtumor-bearing mouse according to the literature procedures. (Refs. 32,33). The radioactivity of each sample was analyzed by HPLC and therepresentative radio-HPLC profiles were shown in FIG. 20A and FIG. 20B.The retention time of free ⁶⁴Cu²⁺ was around. 3.0 min and the⁶⁴Cu-AmBaSar-RGD complex was around 15˜16 min (FIG. 20A). The amount ofintact tracer in the blood, tumor, liver, and kidneys were approximately88%, 95%, 98%, and 98% for ⁶⁴Cu-AmBaSar-RGD (FIG. 20A) and 38%, 87%,34%, and 74% for ⁶⁴Cu-DOTA-RGD (FIG. 20B) at 1 h post injection,respectively.

These experiments demonstrated the ⁶⁴Cu-complexing moiety, AmBaSar is asuperior ligand for an imaging application and targeted radiotherapy dueto its improved stability compared with DOTA. The new PET tracer⁶⁴Cu-AmBaSar-RGD can be used for imaging integrin α_(v)β₃ expression.Our studies demonstrate that ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD havecomparable in vitro stability, lipophilicity, and tumor uptake. However,⁶⁴Cu-AmBaSar-RGD did demonstrate improved in vivo stability andbiodistribution pattern compared with ⁶⁴Cu-DOTA-RGD.

For radiochemistry, the ⁶⁴Cu²⁺ labeling condition of AmBaSar-RGD wasmore favorable and the ⁶⁴Cu-AmBaSar-RGD could be obtained with higherradiochemical yield (≥95%) and purity (≥99%) under mild conditions (pH5.0˜5.5, 23˜37° C.) in less than 30 min, compared with 90% radiochemicalyield for ⁶⁴Cu-DOTA-RGD after incubation at 45° C. for 45 min.

Under in vitro conditions, both ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD werevery stable in PBS, FBS, and mouse serum solutions at physiologicaltemperature. The Log P is a very useful parameter that can be used tounderstand the behavior of drug molecules and predict the distributionof a drug compound in a biological system in combination with the otherparameters. (Ref. 34). The log P values of ⁶⁴Cu-AmBaSar-RGD and⁶⁴Cu-DOTA-RGD indicated that both tracers are rather hydrophilic andthey should show rapid blood clearance and preferential renal excretionif they are stable in vivo. However, compared with ⁶⁴Cu-AmBaSar-RGD,⁶⁴Cu-DOTA-RGD had significantly higher liver uptake (˜2-3 times) basedon microPET imaging and biodistribution studies, which indicated that⁶⁴Cu-DOTA-RGD is less stable than ⁶⁴Cu-AmBaSar-RGD in vivo. In microPETstudies, the U87MG tumor uptake of ⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGDwere comparable at selected time points, and the binding specificity wasproven by the blocking experiment. The biodistribution study wasconsistent with the microPET studies. Similar tumor uptake of bothtracers may be attributed to their comparable hydrophilicity and theminimal impact of chelators on the integrin binding affinity of RGDpeptides. (Ref. 35) ⁶⁴Cu-AmBaSar-RGD cleared rapidly from the blood andpredominantly through the kidneys and ⁶⁴Cu-DOTA-RGD mainly clearedthrough both kidneys and liver. This difference might because that⁶⁴Cu-AmBaSar-RGD is more stable in vivo compared with ⁶⁴Cu-DOTA-RGD,which would result in reduced nonspecific binding. To further confirmthis statement, we also studied the metabolic stability of⁶⁴Cu-AmBaSar-RGD and ⁶⁴Cu-DOTA-RGD in blood, liver, kidneys, and tumorin nude mice bearing U87MG glioma xenografts after 1 h p.i. injection.Our study clearly demonstrated that the amount of intact⁶⁴Cu-AmBaSar-RGD was much higher than that of ⁶⁴Cu-DOTA-RGD in blood,tumor, liver, and kidneys. To the best of our knowledge, this is thefirst study that directly demonstrates that Sar-type chelator, such asAmBaSar, forms more a stable Cu complex in vivo than the establishedchelator DOTA through direct comparison of their metabolic stability.The rapid renal clearance of ⁶⁴Cu-AmBaSar-RGD, as opposed to the mixedrenal and hepatic clearance of ⁶⁴Cu-DOTA-RGD, is preferred as it willprovide better somatic contrast from the imaging perspective, andpotentially reduce radiation exposure.

Although the invention has been described and explained in terms ofspecific embodiments and especially embodiments related to the specificBFC AmBaSar, it should be understood that the present invention is notlimited to the embodiments specifically discussed and the specificembodiments discussed herein are simply illustrative. As will beappreciated by those skilled in the art, the data and informationgleaned from the embodiments discussed with respect to AmBaSar areapplicable to the disclosed BFCs not specifically discussed throughoutthe application.

The following references are incorporated herein by reference in theirentirety.

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We claim:
 1. A chemical composition comprising a molecule selected fromthe group consisting of

wherein the molecule is complexed with a metal or metal ion.
 2. Thechemical composition according to claim 1, wherein the metal is selectedfrom the group consisting of ⁹⁰Y, Gd, ⁶⁸Ga, ⁵⁷Co, ⁶⁰Co, ⁵²Fe, ⁶⁴Cu and⁶⁷Cu.
 3. A bifunctional chelator and targeting moiety conjugatecomprising a molecule selected from the group consisting of

wherein the targeting moiety is selected from the group consisting of apeptide and an antibody.
 4. The bifunctional chelator and targetingmoiety conjugate according to claim 3, wherein the peptide is selectedfrom the group consisting of a RGD, Asp-Gly-Glu-Ala (DGEA), bombesinpeptides (BBN), and uPAR peptides.
 5. A method of positron emissiontomography (PET) imaging comprising: introducing into a subject achemical composition according to claim 2; and imaging the subject afterintroducing the composition.